Confocal microscopy is now established as a valuable tool for obtaining high resolution images and 3-D reconstructions of a variety of biological specimens. In particular, confocal microscopy has the capability to quickly provide information about the biochemical and morphological changes that occur as tissue becomes neoplastic.
In reflectance confocal microscopy, a laser light beam is expanded to make optimal use of the optics in the objective. Through an X-Y deflection mechanism, the laser beam is turned into a scanning beam, focused to a small excitation spot by an objective lens onto a specimen. Reflected light is captured by the same objective and, after conversion into a static beam by the X-Y deflection mechanism, is focused onto a photodetector. A confocal aperture (e.g., a pinhole) is placed in front of the photodetector, such that the reflected light from points on the specimen that are not within the focal plane (the so called "out-of-focus" light) where the laser beam was focused will be largely obstructed by the pinhole. In this way, out-of-focus information (both above and below the focal plane) is greatly reduced. This becomes especially important when dealing with thick specimens. The spot that is focused on the center of the pinhole is often referred to as the "confocal spot."
A 2-D image of a small partial volume of the specimen centered around the focal plane (referred to as an optical section) is generated by performing a raster sweep of the specimen at that focal plane. As the laser scans across the specimen, the analog light signal, detected by the photodetector, is converted into a digital signal, contributing to a pixel-based image displayed on a computer monitor attached to the confocal microscope. The relative intensity of the light reflected from the laser "hit" point, corresponds to the intensity of the resulting pixel in the image (typically 8-bit grayscale). The plane of focus (Z-plane) is selected by a computer-controlled fine-stepping motor which moves the microscope stage up and down. Typical focus motors can adjust the focal plane in as little as 0.1 micron increments. A 3-D reconstruction of a specimen can be generated by stacking 2-D optical sections collected in series.
High resolution confocal imaging can be used to obtain near real-time reflected light images of human epithelial tissue in vivo with micron resolution. In vivo confocal imaging can provide information about subcellular morphologic and biochemical changes in epithelial cells which may be useful in the recognition and monitoring of epithelial precancers in organ sites such as the uterine cervix and oral mucosa. Much of the work demonstrating the potential of confocal microscopy to image cell morphology has been carried out in pigmented tissue where melanin within cells provided the confocal signal and image contrast. More recent work has demonstrated confocal microscopy has the ability to visualize structure in amelanotic cells as well. However, the level of native contrast between diagnostically important structures such as the nucleus and the remainder of the cell's contents can vary significantly among cell types due to differences in cell composition.
Indeed, a problem with most optical examination systems and techniques, including reflectance confocal microscopy, is obtaining suitable signals indicative of the property to be measured. Contrast agents have been commonly applied to tissue in vitro and in vivo to enhance the optical return signal of illuminated tissue and thus aid in the extraction of diagnostically useful information from the sample. For example, techniques are commonly used to highlight cellular structures when using light microscopy to examine tissue samples. On a more gross level, sensitive differentiation between normal tissue and neoplasia in various tissue sites has been recently demonstrated through the use of 5-aminolevulinic acid induced protoporphyrin IX fluorescence.
Acetic acid is routinely used during colposcopy, a procedure involving examination of the cervix in situ with a low power microscope, to enhance differences between normal and diseased regions of the cervical epithelium. Areas which may develop into cervical cancer undergo a transient whitening (acetowhitening) visible to the naked eye. While the mechanism behind this phenomenon is not yet fully understood, it is commonly agreed that the higher nuclear density present in abnormal epithelium is a significant factor.
The inventors have determined that it would be desirable to provide a technique for by enhancing the return optical signal for a reflectance confocal microscopy system in a manner that enhances the detection of abnormal tissue. The present invention provides such a technique.